Method of preparing alpha-1-proteinase inhibitor

ABSTRACT

A method is disclosed for separating alpha-1-proteinase inhibitor from a blood plasma fraction particularly from the Cohn fractionation scheme. An aqueous solution of the blood plasma fraction is held at a pH of about 6.5-8.5, and a temperature of about 2°-50° C. for a period of about 0.2-24 hours and then mixed with a poly-condensed polyglycol. The mixture may be held at a temperature of about 2°-10° C. for a period of about 1-24 hours. Next, the pH of the mixture is adjusted to selectively precipitate unwanted proteins from the solution of alpha-1-proteinase inhibitor which is separated from the solution and purified further. The amount of poly-condensed polyglycol and the pH of the mixture are selected accordingly.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of co-pending application U.S. Ser. No.389,202, filed June 17, 1982, now U.S. Pat. No. 4,379,087.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to and has among its objects the provision of anovel method for separating alpha-1-proteinase inhibitor (PI) from bloodplasma or blood plasma fractions. Further objects of the invention willbe evident from the following description wherein parts and percentagesare by weight unless otherwise specified.

2. Description of the Prior Art

Alpha-1-proteinase inhibitor is a glycoprotein having molecular weightof 54,000. The protein consists of a single polypeptide chain to whichseveral oligosaccharide units are covalently bound. Human PI has a rolein controlling tissue destruction by endogenous serine proteinases. Agenetic deficiency of PI, which accounts for 90% of the trypsininhibitory capacity in blood plasma, has been shown to be associatedwith premature development of pulmonary emphysema. The degradation ofelastin associated with emphysema probably results from a localimbalance of elastolytic enzymes and the naturally occurring tissue andplasma proteinase inhibitors. PI rapidly inhibits human pancreatic andleukocyte elastases (Biochem. Biophys. Res. Comm., Vol. 72, No. 1, pages33-39, 1976; ibid., Vol. 88, No. 2, pages 346-350, 1979).

A number of methods have been employed to isolate PI from the bloodplasma. A majority of these methods are directed to laboratory scaleisolation while others pertain to production on a commercial level.

Pannell et al., Biochemistry, Vol. 13, pages 5439-5445, (1974), employeda process wherein albumin-poor blood plasma was pooled and fractionatedwith solid ammonium sulfate (0.60-0.80 saturation). The precipitateresulting was solubilized and dialyzed and applied to a column ofDEAE-cellulose. The 0.05-0.15 M NaCl linear gradient is pooled,concentrated, and dialyzed, and then applied again to a column ofDEAE-cellulose. The linear gradient from 0.05-0.20 NaCl was collected,pooled, and concentrated to give PI.

In the method of Saklatvala et al., Biochem. J., Vol. 157, pages 339-351(1976), human plasma was fractionated using ammonium sulfate (80%saturation) to give a precipitate, which was dissolved, dialyzed andchromatographed on DEAE-cellulose. The 0.5 M NaCl extract was applied toa concanavalin A-Sepharose column. The alpha-D-methyl glucopyranosideeluate was concentrated and applied again to a DEAE-cellulose column.The 0.0-0.2 M NaCl eluate contained PI.

Fifty percent saturated ammonium sulfate precipitation was used byMusiani et al., Biochem., Vol. 15, pages 798-804 (1976) to separate aPI-rich fraction that was solubilized and then subjected to successivechromatographic steps using DEAE ion exchanger, concanavalinA-Sepharose, Sephadex G-100, and an immunoadsorbent column to yieldpurified PI.

A large scale purification of PI from human plasma was disclosed byKress et al., Preparative Biochemistry, Vol. 3, No. 6, pages 541-552(1973). The precipitate from the 80% ammonium sulfate treatment of humanplasma was dialyzed and chromatographed on DEAE-cellulose. Theconcentrate obtained was again dialyzed and gel filtered on SephadexG-100. The PI-containing fractions were chromatographed twice on DE-52cellulose to give PI.

Glaser et al., ibid., Vol. 5, No. 4, pages 333-348 (1975) isolated PIfrom Cohn Fraction IV-1 in 30% overall yield. Dissolved IV-1 waschromatographed on DEAE-cellulose, QAE-Sephadex, concanavalinA-Sepharose, and G-150 Sephadex to give PI.

An integrated plasma fractionation system based on polyethylene glycol(PEG) was disclosed by Hao et al., Proceedings of the InternationalWorkshop on Technology for Protein Separation and Improvement of BloodPlasma Fractionation, held Sept. 7-9, 1977, Reston, Va. In the publishedmethod Cohn cryoprecipitate was mixed with PEG in an amount of 40 gramsper liter (g/l). All operations were conducted at 5° C.

After stirring for 60 minutes, the first fraction was removed bycentrifugation. An additional 60 g/l of PEG was added to the supernate(final concentration approximately 10%). Prothrombin complex (PTC) wasthen extracted from the 10% PEG supernate by batch-wise adsorption onDEAE cellulose, and an additional 100 g/l of PEG was added to obtain the10-20% PEG precipitate. The four fractions thus obtained were 0-4% PEGprecipitate, 4-10% PEG precipitate, 10-20% PEG precipitate and 20% PEGsupernate, and were designated as Fractions A, B, C and D, respectively.It should be pointed out that these PEG concentrations were based on theoriginal volume of cryosupernate.

The distribution of proteins in the four PEG fractions was as follows:Fibrinogen was the dominant protein in Fraction A with albumin being themajor contaminant. Most of the contaminating albumin in Fractions A, Band C resulted from coprecipitation and/or entrapment of supernate sincealbumin by itself did not precipitate under these conditions. Fraction Bwas rich in plasminogen, C3 component of complement, IgG and IgM. Inaddition, virtually all of the beta-lipoproteins were present in thisfraction. Fraction C contained appreciable quantities of alpha₂macroglobulin, IgA and was rich in prothrombin and other coagulationfactors which constitute the so-called prothrombin complex. However, theauthors found that better yields of PTC could be obtained from the 10%PEG supernate rather than from the 10-20% PEG precipitate. Fraction Dwas dominated by albumin but also contained all of the alpha-1-acidglycoprotein as well as most of the PI, antithrombin III (AT III),ceruloplasmin (C_(p)), haptoglobin, transferrin (T_(f)) and Cl esteraseinhibitor (Cl inhib.). Several additional proteins were also isolatedfrom Fraction D including prealbumin (PA), retinol binding protein(RBP), transcortin, and angiotensinogen. In general, most of the smallerproteins were in Fraction D.

SUMMARY OF THE INVENTION

The invention described herein is a method for separatingalpha-1-proteinase inhibitor from blood plasma or blood plasma fractionswhich contain PI. In the present method blood plasma is fractionatedaccording to the Cohn ethanol fractionation technique or itsmodifications [see for example, Cohn et al., J. Am. Chem. Soc., 68, 459,(1946); Oncley et al., ibid., 71, 541 (1949); U.S. Pat. No. 2,390,074;"The Plasma Proteins," second edition, Volume III, pages 548-550,Academic Press, New York, N.Y.] to give a Cohn fraction containing PI.An aqueous solution of the PI-containing Cohn fraction is prepared, andthe pH of the solution is adjusted to about 6.5-8.5. Following a holdperiod of about 0.2-24 hours at 2°-50° C., a polycondensed polyglycol,for example, polyethylene glycol (PEG) is added to the solution, the pHof which is then adjusted to selectively precipitate unwanted proteinsfrom the effluent in which a substantial proportion of the PI isretained. The effluent is treated to separate PI therefrom directly orwith further purification.

The primary advantage of the present method is that PI may be separatedfrom blood plasma in high yield. The PI obtained by our method has ahigh specific activity when compared to the product obtained by many ofthe methods of the prior art.

Another advantage of the invention is the ease with which the method maybe applied to the separation of PI from blood plasma. The separation, inaddition, can be carried out inexpensively because of the low cost ofthe agents employed in the present method.

An important feature of the present invention is the discovery that theconcentration of PEG and subsequent pH adjustment employed have a markedeffect on the ability to separate PI from the remaining proteins. Ifabout 20% PEG is used to treat dissolved Fraction IV-1, the PI remainsin solution and precipitates from the PEG supernatant by acidifying topH 5.1-5.5. However, other proteins are also found in the precipitatethat forms. Surprisingly, however, we have discovered that treatment ofFraction IV-1, with an amount of a polycondensed polyglycol at aparticular pH according to FIG. 1 results in a situation wherein the PIdoes not precipitate from the supernatant when the latter is acidified.Other proteins present in the supernatant do precipitate. Separation ofthe precipitate from the supernatant yields a PI-containing supernatantfrom which PI having a specific activity greater than about 0.2, usuallyabout 0.20-0.27, can readily be obtained in yields greater than about60%.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph depicting the relationship of pH and the amount ofpolycondensed polyglycol for achieving the benefits of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As mentioned above, the starting material for the method of theinvention is blood plasma that has been fractionated by the Cohn ethanolfractionation technique or its modifications to give a concentratecontaining PI such as, among others, Cohn Fraction IV and IV-1 andincludes reworks of these fractions wherein other proteins are firstremoved. In the following description emphasis is directed to CohnFraction IV-1 by way of illustration and not limitation.

Fraction IV-1 paste may be used directly or it may first be treated toremove lipids contained therein, for example, by contact with "cold"acetone, fumed silica (Aerosil®, Pacific Coast Chemicals, Emeryville,Calif.), calcium and dextran sulfate, and the like. For instance, IV-1paste may be mixed with acetone in the proportion of about 10-40 partsof acetone per part of Fraction IV-1. The temperature during thistreatment is maintained at about -30° to -35° C., the startingtemperature of the cold acetone. Acetone also removes water fromFraction IV-1 paste, thereby resulting, upon removal of the acetone byconventional means, in a dry powder containing substantially all of thePI.

An aqueous solution or suspension of Fraction IV-1 paste or powder asdescribed above is prepared by mixing Fraction IV-1 with water in theproportion of about 8-12 grams of Fraction IV-1 per 100 ml of water. Atthis time, the pH of the mixture is adjusted to about 6.5-8.5 by theaddition of a physiologically-acceptable agent such astris-(hydroxymethyl) aminomethane (TRIS) and the like. In addition, aphysiologically-acceptable salt such as sodium chloride or the like maybe added to the aqueous mixture in an amount sufficient to achieve aconcentration of about 0.01-0.10 M.

It is within the compass of the invention, and preferred, to mixFraction IV-1 with a buffer of a particular pH so that the resultingmixture will have a pH and a concentration within the ranges quotedabove. As the buffer one may use TRIS hydrochloride and the like.Additionally, the buffer may contain a physiologically-acceptable saltin an amount so that its concentration in the final mixture of FractionIV-1 and buffer will be about 0.01-0.10 M.

The above mixture is prepared at a temperature of about 2°-50° C., andthen held at a temperature within the above range for a period of 0.2-24hours. There is an inverse relationship between the time and temperaturein this step. Generally, for each 10° C. rise in temperature the holdtime should decrease by half with the preferred conditions being about0.5 hour at about 45° C. or about 8 hours at about 5° C.

Following this holding period a polycondensed polyglycol, e.g., PEG, ofmolecular weight of about 2,000-10,000 is added to the mixture. Forpurposes of the invention the amount of polycondensed glycol is afunction of the pH at which the mixture is to be adjusted. Therelationship is set forth in FIG. 1. Thus, for example, about 8-10 gramsof polycondensed polyglycol per ml of aqueous mixture containingFraction IV-1 are employed at pH about 4.6, about 9-12 grams/ml at pHabout 5.0, about 12-14 grams/ml at pH about 5.5, about 14-16 grams/ml atpH about 6.0, about 16-18 grams/ml at pH about 6.5, and about 18-21grams/ml at pH about 7.0. Preferably, about 10-15 grams of polycondensedglycol are used per 100 ml of the aqueous mixture containing FractionIV-1 with pH of about 4.6-5.7. Usually, the amount of polycondensedglycol employed is determined by the initial amount of Fraction IV-1paste mixed with water in the initial step in this method. Thus, if 8grams of Fraction IV-1 were mixed with 100 ml of water, then about 10-11g of polycondensed polyglycol should be used per 100 ml of aqueousmixture. On the other hand if 10 grams of Fraction IV-1 were initiallymixed with 100 ml of water, then about 14-15 g of polycondensedpolyglycol per 100 ml of aqueous mixture should be employed in thisstep. The relationship, therefore, between the initial amount ofFraction IV-1 and the polycondensed polyglycol is about 1 gram of glycolper about 0.5-1.0 grams of Fraction IV-1. The more preferable agents andamounts in this particular step are about 11 grams of a PEG having amolecular weight of about 3,000-4,000 and about 8 grams of Fraction IV-1per 100 ml of aqueous mixture.

The mixture of polycondensed polyglycol and Fraction IV-1 may be heldfor a period of about 1-24 hours at a temperature of about 2°-10° C.

The precipitate that forms can be separated by conventional means suchas centrifugation and discarded, and the effluent can be treated asdescribed hereinbelow. However, it is preferred not to separate theeffluent and precipitate at this time. Rather, the pH of the entiremixture is adjusted to a value determined by reference to FIG. 1. In thepreferred instance wherein about 10-15 grams of polycondensed polyglycolare used per ml of mixture the pH is adjusted to within the range ofabout 4.6-5.7 by the addition thereto of a physiologically-acceptableacid such as acetic acid, hydrochloric acid, citric acid, phosphoricacid, or the like. The acidified mixture is held for only 0-60 minutes,or generally, for as short a time as possible since the yield of activePI decreases with time. By selection of the amount of polycondensedpolyglycol and pH according to FIG. 1, the PI can be maintained insolution with precipitation of unwanted proteins. The precipitate thatforms containing unwanted proteins is separated from the solution, againby conventional means such as centrifugation, and discarded.

The pH of the remaining solution is adjusted to about 5.5-8.6,preferably about 6.5, by the addition of a physiologically acceptablealkaline material such as, for example, sodium hydroxide.

The so-adjusted material is then contacted with an anion exchange mediumsuch as DEAE-Sephadex, QAE-Sephadex, DEAE-Sephacel, DEAE-cellulose,DEAE-Sepharose or the like. A variety of conditions may be used in thisparticular step. Contact with the above agent may be carried outbatch-wise or continuously. For best results the anion exchange mediumis placed in a chromatographic column and the PI eluted therefrom. Ingeneral, the anion exchange medium is first equilibrated in a buffersolution of pH about 5.5-8.6. Next, the anion exchange medium iscontacted with the above solution containing PI in the proportion ofabout 10-15 volumes of solution of 1 volume exchanger. The anionexchange medium is washed again with a buffer solution, usually the samebuffer solution as above; the amount of this wash solution generally isabout 3-10 volumes per volume of exchanger.

The PI is removed by either gradient elution or stepwise elution fromthe anion exchange medium by contacting it with a buffer solution of pHabout 5.5-8.6 containing 0.0-0.3 M sodium chloride, 0.01-0.12 M disodiumphosphate, and the like or combinations thereof.

Alternative to the use of an anion exchange medium, PI may be separatedfrom the solution after adjustment to pH 4.6-5.4 by the addition of PEGin the amount of 10-30 g per 100 ml of solution. A precipitatecontaining PI is separated from the solution.

Following the separation of the solution containing PI, for example,from the anion exchange medium, the solution is treated to reduce itswater content and change the ionic composition by conventional meanssuch as by diafiltration, ultrafiltration, lyophilization, etc., orcombinations thereof.

The PI concentrates can be formulated into pharmaceutical preparationsfor therapeutic, diagnostic, or other uses. To prepare them forintravenous administration the compositions are dissolved usually inwater containing physiologically compatible substances such as sodiumchloride, glycine, and the like and having a buffered pH compatible withphysiological conditions. Generally, guidelines for intravenouslyadministered compositions are established by governmental regulations.

It is desirable that the PI concentrates be non-hepatitis infective. Inthis respect The concentrates may be treated to reduce hepatitisinfectivity by, for example, pasteurization, i.e., heating at atemperature and for a time, such as, for example, at about 60° C. ormore for a period up to about 10 hours, sufficient to render the PIhepatitis non-infective. To stabilize the PI during this heat treatmenta source of citrate ions is added in an amount sufficient to stabilizethe PI during heating. Generally, if about 20 mg of total protein ispresent in the PI concentrate, then the solution is made about 0.25-0.5M in citrate ion. The pH of the mixture during this heating step shouldpreferably be about 6.0-7.0.

To achieve maximum stabilization of PI during heating it is desirable touse a carbohydrate as the stabilization agent either alone or withsodium citrate. For this purpose one may use as the carbohydrate amono-, di-, and trisaccharide such as arabinose, glucose, galactose,maltose, fructose, fibose, mannose, rhammose, sucrose, etc., or a sugaralcohol such as sorbitol and mannitol, etc., in an amount of about0.5-2.4 g/ml of PI solution.

As mentioned above the pasteurized products of the invention may beincorporated into pharmaceutical preparations which may be used fortherapeutic purposes. However, the term "pharmaceutical preparation" isintended in a broader sense herein to include preparations containing aprotein composition in accordance with this invention used not only fortherapeutic purposes, but also for reagent or diagnostic purposes asknown in the art or for tissue culture. The pharmaceutical preparationintended for therapeutic use should contain a therapeutic amount of PI,i.e., that amount necessary for preventative or curative healthmeasures. If the pharmaceutical preparation is to be employed as areagent or diagnostic, then it should contain reagent or diagnosticamounts of PI.

EXAMPLES

The invention described above is demonstrated further by the followingillustrative examples.

ASSAYS

PI is estimated by its elastase inhibitory capacity, using a chromogenicsubstrate for elastase. Hydrolysis ofN-succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide (SA₃ pNA) byelastase causes an increase in absorption at 405 nm. This increase iscontinuously monitored usually at 37° C. Comparisons of the linearchanges of absorbance with time in the presence and absence of sample(PI) are made. The amount of inhibitor is then calculated based on theknown molecular weights of elastase and PI, on the known 1:1stoichiometry, and on the known amount of elastase used.

PI may also be estimated by its trypsin inhibitory capacity in a similarmanner.

EXAMPLE 1

Fraction IV-1 was obtained by means of the Cohn fractionation scheme asmentioned above.

Fraction IV-1 paste (10.0 g) was dissolved in 0.1 liter of a buffersolution of pH 8.5 containing 0.1 M (TRIS) and 0.02 M sodium chloride.The mixture was stirred for 16 hours at 5° C.

PEG 4000 (from Union Carbide Corporation) was added to a level of 14%(w/v). The mixture was stirred to dissolve the PEG and then centrifugedat 15,000×g. The supernatant was collected, diluted with 0.22 volumes ofwater, acidified to pH 5.1 by addition of 1 N acetic acid, andcentrifuged. The supernatant containing the PI was collected andanalyzed by the aforedescribed methods. The results are found in Table1.

                  TABLE 1                                                         ______________________________________                                                                       Total Re-   Puri-                                             PI       Volume PI    covery                                                                              fication                           Sample  A.sub.280                                                                            (mg/ml)  (ml)   (mg).sup.1                                                                          (mg)  Factor                             ______________________________________                                        Fraction                                                                              30.3   2.0      110    220   100   1.0                                IV-1                                                                          solution                                                                      PEG 4000                                                                              12.6   1.9      110    209   95    2.2                                supernatant                                                                   Acid     4.0   1.4      129    180   82    5.2                                supernatant                                                                   ______________________________________                                    

EXAMPLE 2

The procedure of Example 1 was followed with the exception that theFraction IV-1 in buffer solution was held for varying times prior toaddition of PEG 4000. The results are tabularized below.

                  TABLE 2                                                         ______________________________________                                                                PI in 14% PEG 4000                                    Holding Period                                                                             PI in Solution                                                                           Supernatant                                           (hr.)        (mg/ml)    (mg/ml)                                               ______________________________________                                        0.25         1.9        0.9                                                   1            1.9        1.0                                                   2            1.9        1.3                                                   4            1.9        1.4                                                   7            1.9        1.6                                                   23           1.9        1.6                                                   ______________________________________                                    

EXAMPLE 3

Fraction IV-1 (50 g) was dissolved in 500 ml of 0.1 M Tris buffer, pH7.5, to which was added PEG 4000 to a level of 14% (w/v). The sampleswere centrifuged and the supernatants divided into four equal parts.Each part was diluted with 0.22 volumes of 0.1 M Tris buffer containingvarying amounts of sodium chloride so that the final sodium chlorideconcentrations were 0, 0.01, 0.02, and 0.03 M. Each sample was thenacidified to pH 5.2 by addition of 1 N acetic acid. The samples werecentrifuged, and the supernatants were separated and adjusted to pH 7.4by addition of alkali (1.0 M NaOH).

The results are tabularized below.

                  TABLE 3                                                         ______________________________________                                                          Volume   PI     Total PI                                                                             Yield                                Sample     A.sub.280                                                                            (ml)     (mg/ml)                                                                              (mg)   (%)                                  ______________________________________                                        IV-1 solution                                                                            31.3   545      1.92   1037   100                                  PEG supernatant                                                                          13.2   570      1.36   762    73                                   Acid supernatant                                                                         4.27   1.70     0.67   113    60                                   0.03 M NaCl                                                                   Acid supernatant                                                                         4.02   170      0.71   121    64                                   0.02 M NaCl                                                                   Acid supernatant                                                                         3.94   170      0.61   104    55                                   0.01 M NaCl                                                                   Acid supernatant                                                                         4.00   170      0.60   102    54                                   0.00 M NaCl                                                                   ______________________________________                                    

EXAMPLE 4

PI acid supernatant was prepared according to the procedure of Example1; 1300 ml of this supernatant was combined with 650 ml of water andplaced on a 100 ml column of DEAE-Sephacel equilibrated with 0.01 Msodium phosphate at pH 6.5. After the column was washed with one columnvolume of 0.01 M sodium phosphate, the column was eluted with a lineargradient to 0.12 M sodium chloride (total volume of wash was 1.5 l). Theactive fractions were combined.

The above manipulations were repeated using DEAE-Sepharose as the anionexchanger medium in place of DEAE-Sephacel and also repeated using 0.12M disodium phosphate as the eluting medium. The results are summarizedbelow.

                  TABLE 4                                                         ______________________________________                                        Anion                   Specific    PI                                        Exchange                Activity    Yield                                     Medium   Eluting Medium (mg/ml/A.sub.280)                                                                         (%)                                       ______________________________________                                        DEAE-    sodium chloride                                                                              0.98        64                                        Sephacel                                                                      DEAE-    sodium chloride                                                                              1.12        63                                        Sepharose                                                                     DEAE-    disodium phosphate                                                                           1.30        83                                        Sepharose                                                                     ______________________________________                                    

EXAMPLE 5

PI acid supernatant was prepared according to the procedure of Example1; 1300 ml of this supernatant was combined with 650 mlwater-for-injection (WFI), and 100 ml of anion exchange medium, andbuffered with 0.01 M sodium phosphate, 0.05 M sodium chloride, pH 6.5.The mixture was stirred for 1 hour and allowed to settle. Afterfiltration, the medium was washed five times with 250 ml of the abovebuffer and then was eluted by treatment with 250 ml of the above bufferat 0.15 M sodium chloride (five washes). The results are summarizedbelow.

                  TABLE 5                                                         ______________________________________                                                        PI Yield                                                      Anion Exchange Medium                                                                         (%)       Purification Factor                                 ______________________________________                                        DEAE-Sephadex A-50                                                                            80        2.1                                                 Whatman DE-52 Cellulose                                                                       44        1.8                                                 QAE-Sephadex    71        1.6                                                 Phoenix DEAE-Cellulose                                                                        55        1.8                                                 ______________________________________                                    

EXAMPLE 6

PI supernatant was prepared according to the procedure of Example 5 andwas concentrated by means of hollow fiber ultrafiltration to aconcentration of 10 mg PI/ml of supernatant. The pH was adjusted to 7 to6.5. Aliquots of this concentrated material were taken and mixed with astabilizer or not as summarized in the Table below. The aliquots werethen heated at 60° C. for 10 hours. The results appear in the followingTable.

                  TABLE 6                                                         ______________________________________                                                                 PI                                                   Sample             pH    % Recovery                                           ______________________________________                                        0.5 M sodium citrate                                                                             7     58                                                   0.5 M sodium citrate                                                                             6.5   90                                                   1.2 g/ml sucrose   7     100                                                  0.5 M sodium citrate +                                                                           6.5   100                                                  0.8 g/ml sucrose                                                              no stabilizer      7     12                                                   ______________________________________                                    

We claim:
 1. A method for separating alpha-1-proteinase inhibitor from ablood plasma fraction containing the same, which comprises the stepsof(a) holding an aqueous solution of the blood plasma fraction at a pHof about 6.5-8.5, and a temperature of about 2°-50° C. for a period ofabout 0.2-24 hours, (b) mixing the solution with a polycondensedpolyglycol and adjusting the pH of the mixture to selectivelyprecipitate unwanted proteins from the solution without precipitation ofalpha-1-proteinase inhibitor, the amount of polycondensed polyglycol andthe pH of the mixture being determined according to FIG. 1, and (c)separating alpha-1-proteinase inhibitor from the solution.
 2. The methodof claim 1 wherein 1 part of polyglycol is used per 0.5-1.0 parts ofblood plasma fraction.
 3. The method of claim 1 wherein the mixture ofthe solution with the polyglycol is held at a temperature of about2°-10° C. for a period of about 1-24 hours prior to the adjustment ofthe pH of the mixture.
 4. The method of claim 1 which further includesthe step of treating the alpha-1-proteinase inhibitor to render ithepatitis non-infective.
 5. The method of claim 4 wherein thealpha-1-proteinase inhibitor is rendered hepatitis non-infective byheating an aqueous solution of the alpha-1-proteinase inhibitor in thepresence of a heat stabilizing agent for a time and at a temperature andpH sufficient to render the alpha-1-proteinase inhibitor hepatitisnon-infective.
 6. The method of claim 5 wherein the heat stabilizingagent is a source of citrate ions.
 7. The method of claim 5 wherein theheat stabilizing agent is a carbohydrate.
 8. The method of claim 5wherein the heat stabilizing agent is a mixture of a carbohydrate and asource of citrate ions.
 9. The method of claim 5 wherein thealpha-1-proteinase inhibitor is heated for about 60° C. or more for aperiod up to about 10 hours at a pH of about 6.0-7.0.
 10. The method ofclaim 1 wherein an effluent solution is separated from a precipitate inStep b prior to adjusting the pH of the mixture.
 11. The method of claim1 wherein the alpha-1-proteinase inhibitor is separated from thesolution in step (c) by(d) separating a supernatant solution containingalpha-1-proteinase inhibitor from a precipitate, (e) contacting thesolution with an anion exchange medium at a pH of about 5.5-8.6, and (f)selectively separating an effluent containing alpha-1-proteinaseinhibitor from the anion exchange medium.
 12. The method of claim 11wherein the effluent of step (f) is treated to reduce its water content.13. The method of claim 1 wherein the blood plasma fraction containingalpha-1-proteinase inhibitor is selected from the group consisting ofCohn Fraction IV-1, Cohn Fraction IV, and their reworks.
 14. The methodof claim 1 wherein the alpha-1-proteinase inhibitor is separated fromthe mixture in step (c) by contacting the mixture with a polycondensedpolyglycol and separating an effluent from a precipitate containingalpha-1-proteinase inhibitor.
 15. The method of claim 1 wherein thepolycondensed polyglycol is polyethylene glycol.
 16. The method of claim15 wherein the molecular weight of the polyethylene glycol is about3000-4000.
 17. The method of claim 1 wherein the blood plasma fractionis treated with fumed silica to remove lipids contained therein.
 18. Amethod for removing lipids from Fraction IV, Fraction IV-1, or theirreworks which comprises contacting said fraction with fumed silica. 19.A method for separating alpha-1-proteinase inhibitor from a blood plasmafraction containing the same, which comprises the steps of(a) treatingthe blood plasma fraction containing alpha-1-proteinase to remove lipidscontained therein, (b) holding an aqueous solution of the blood plasmafraction at a pH of about 6.5-8.5, and a temperature of about 2°-50° C.for a period of about 0.2-24 hours, (c) mixing the solution with apolycondensed polyglycol and adjusting the pH of the mixture toselectively precipitate unwanted proteins from the solution withoutprecipitation of alpha-1-proteinase inhibitor, the amount ofpolycondensed polyglycol and the pH of the mixture being determinedaccording to FIG. 1, and (d) separating alpha-1-proteinase inhibitorfrom the solution.